Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

The process in which a signal is passed on to downstream components within the cell, which become activated themselves to further propagate the signal and finally trigger a change in the function or state of the cell.

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.

Keywords

Protein which is part of a reference proteome. Reference proteomes are a subset of proteomes that have been selected either manually or algorithmically according to a number of criteria to provide a broad coverage of the tree of life and a representative cross-section of the taxonomic diversity found within UniProtKB, as well as the proteomes of well-studied model organisms and other species of interest for biomedical research.